Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

ABSTRACT

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from  Alicyclobacillus acidocaldarius  and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan, or mannan-decorating groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/587,229, filed Oct. 2, 2009, pending, which is a continuation-in-partof U.S. patent application Ser. No. 12/322,359, filed Jan. 29, 2009, nowU.S. Pat. No. 7,858,353, issued Dec. 28, 2010, to Thompson et al., whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/025,136 filed Jan. 31, 2008, for “THERMOPHILIC AND THERMOACIDOPHILICBIOPOLYMER-DEGRADING GENES AND ENZYMES FROM ALICYCLOBACILLUSACIDOCALDARIUS AND RELATED ORGANISMS, METHODS;” is acontinuation-in-part of U.S. patent application Ser. No. 12/380,551,filed Feb. 26, 2009, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/032,339 filed Feb. 28, 2008, for “THERMOPHILICAND THERMOACIDOPHILIC METABOLISM GENES AND ENZYMES FROM ALICYCLOBACILLUSACIDOCALDARIUS AND RELATED ORGANISMS, METHODS;” is acontinuation-in-part of U.S. patent application Ser. No. 12/380,450,filed Feb. 26, 2009, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/031,984 filed Feb. 27, 2008, for “THERMOPHILICAND THERMOACIDOPHILIC GLYCOSYLATION GENES AND ENZYMES FROMALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS;” is acontinuation-in-part of U.S. patent application Ser. No. 12/380,554,filed Feb. 26, 2009, now U.S. Pat. No. 7,960,534, issued Jun. 14, 2011,to Thompson et al., which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/031,593 filed Feb. 26, 2008, for “THERMOPHILICAND THERMOACIDOPHILIC SUGAR TRANSPORTING GENES AND ENZYMES FROMALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS;” is acontinuation-in-part of U.S. patent application Ser. No. 12/380,008,filed Feb. 20, 2009, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/030,820 filed Feb. 22, 2008, for“TRANSCRIPTIONAL CONTROL IN ALICYCLOBACILLUS ACIDOCALDARIUS ANDASSCIATED GENES, PROTEINS, AND METHODS;” and is a continuation-in-partof U.S. patent application Ser. No. 12/321,636, filed Jan. 23, 2009, nowU.S. Pat. No. 7,923,234, issued Apr. 12, 2011, to Thompson et al., whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/023,639 filed Jan. 25, 2008, for “THERMAL AND ACID TOLERANTBETA-XYLOSIDASES, GENES ENCODING, RELATED ORGANISMS, AND METHODS;” thedisclosure of each of which is hereby incorporated herein by thisreference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under Contract NumberDE-AC07-991D13727 and Contract Number DE-AC07-051D14517 awarded by theUnited States Department of Energy. The government has certain rights inthe invention.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submittedconcomitant with this application, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Dilute acid hydrolysis to remove hemicellulose from lignocellulosicmaterials is one of the most developed pretreatment techniques forlignocellulose and is currently favored (Hemelinck et al., 2005) becauseit results in fairly high yields of xylose (75-90%). Conditions that aretypically used range from 0.1 to 1.5% sulfuric acid and temperaturesabove 160° C. The high temperatures used result in significant levels ofthermal decomposition products that inhibit subsequent microbialfermentations (Lavarack et al., 2002). High temperature hydrolysisrequires pressurized systems, steam generation, and corrosion resistantmaterials in reactor construction due to the more corrosive nature ofacid at elevated temperatures.

Lower temperature acid hydrolyses are of interest because they have thepotential to overcome several of the above shortcomings (Tsao et al.,1987). It has been demonstrated that 90% of hemicellulose can besolubilized as oligomers in a few hours of acid treatment in thetemperature range of 80-100° C. It has also been demonstrated that thesugars produced in low temperature acid hydrolysis are stable underthose same conditions for at least 24 hours with no detectabledegradation to furfural decomposition products. Finally, sulfuric acidtypically used in pretreatments is not as corrosive at lowertemperatures. The use of lower temperature acid pretreatments requiresmuch longer reaction times to achieve acceptable levels of hydrolysis.Although 90% hemicellulose solubilization has been shown (Tsao, 1987),the bulk of the sugars are in the form of oligomers and are not in themonomeric form. The organisms currently favored in subsequentfermentation steps cannot utilize sugar oligomers (Garrote et al., 2001)and the oligomer-containing hydrolysates require further processing tomonomers, usually as a second acid or alkaline hydrolysis step (Garroteet al., 2001).

Other acidic pretreatment methods include autohydrolysis and hot waterwashing. In autohydrolysis, biomass is treated with steam at hightemperatures (˜240° C.), which cleaves acetyl side chains associatedwith hemicellulose to produce acetic acid that functions in a similarmanner to sulfuric acid in acid hydrolysis. Higher pretreatmenttemperatures are required as compared to dilute sulfuric acid hydrolysisbecause acetic acid is a much weaker acid than sulfuric. At temperaturesbelow 240° C., the hemicellulose is not completely hydrolyzed to sugarmonomers and has high levels of oligomers (Garrote et al., 2001). In hotwater washing, biomass is contacted with water (under pressure) atelevated temperatures 160-220° C. This process can effectively hydrolyzegreater than 90% of the hemicellulose present, and the solubilizedhemicellulose was typically over 95% in the form of oligomers (Liu andWyman, 2003).

BRIEF SUMMARY OF THE INVENTION

The entire contents of each of the following applications areincorporated herein in their entirety by this reference: patentapplication Ser. Nos. 12/322,359 (filed Jan. 29, 2009); 12/321,636(filed Jan. 23, 2009); 12/380,008 (filed Feb. 20, 2009); 12/380,554(filed Feb. 26, 2009); 12/380,450 (filed Feb. 26, 2009); and 12/380,551(filed Feb. 26, 2009).

Embodiments of the invention relate to a genetically modified organismfor converting biomass into products. The genetically modified organismmay comprise at least one nucleic acid sequence encoding a polypeptideas disclosed in the patent applications previously incorporated byreference herein. In embodiments, the genetically modified organism maycomprise at least one nucleic acid sequence encoding a polypeptideassociated with at least partially degrading, cleaving, transporting,metabolizing and/or removing polysaccharides, cellulose, hemicellulose,lignin, starch, sugars, sugar oligomers, carbohydrates, complexcarbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-,galactan, or mannan-decorating groups; and/or at least one nucleic acidsequence encoding a polypeptide associated with fermenting sugarmolecules to products.

Embodiments of the invention also relate to protein(s) and/or cellularextracts isolated from a genetically modified organism. The isolatedprotein and/or cellular extracts may comprise: at least one polypeptideisolated from a genetically modified organism, the organism including:at least one recombinant nucleic acid encoding a polypeptide asdisclosed in the patent applications previously incorporated byreference herein. In embodiments, the at least one recombinant nucleicacid encoding a polypeptide may comprise: at least one polypeptideinvolved in at least partially degrading, cleaving, transporting,metabolizing and/or removing polysaccharides, cellulose, hemicellulose,lignin, starch, sugars, sugar oligomers, carbohydrates, complexcarbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-,galactan, or mannan-decorating groups; and/or at least one recombinantnucleic acid encoding a polypeptide involved in fermenting sugarmolecules to products.

Additional embodiments of the invention relate to methods of at leastpartially processing polysaccharides, cellulose, hemicellulose, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycosides, xylan-, glucan-, galactan, ormannan-decorating groups into a product. The method may comprise:placing a genetically modified organism in fluid contact with apolysaccharide, cellulose, hemicellulose, starch, sugars, sugaroligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans,glycoside, xylan-, glucan-, galactan, and/or mannan-decorating group.The genetically modified organism may comprise at least one nucleic acidand/or at least one recombinant nucleic acid encoding a polypeptideinvolved in at least partially degrading, cleaving, transporting,metabolizing, or removing polysaccharides, cellulose, hemicellulose,lignin, starch, sugars, sugar oligomers, carbohydrates, complexcarbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-,galactan, or mannan-decorating groups; and/or at least one nucleic acidand/or at least one recombinant nucleic acid encoding a functionalprotein involved in fermenting sugar molecules to a product and/or afermentation enzyme involved in converting sugars into a product.

Further embodiments of the invention relate to isolating an extract froma genetically modified organism according to the present invention.Embodiments may also include placing the isolated extract in fluidcontact with a polysaccharide, cellulose, hemicellulose, lignin, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycoside, xylan-, glucan-, galactan, and/ormannan-decorating group. The genetically modified organism may compriseat least one nucleic acid and/or at least one recombinant nucleic acidencoding a polypeptide having some level of activity in at leastpartially degrading, cleaving, transporting, metabolizing, and/orremoving polysaccharides, cellulose, hemicellulose, lignin, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycosides, xylan-, glucan-, galactan, ormannan-decorating groups; and/or at least one nucleic acid and/or atleast one recombinant nucleic acid encoding a functional proteininvolved in fermenting sugar molecules to a product such as afermentation or other enzyme involved in converting sugars into aproduct.

DETAILED DESCRIPTION OF THE INVENTION

Lignocellulose is a highly heterogeneous 3-dimensional matrix comprisedof cellulose, hemicellulose, and lignin. Many fuels and chemicals can bemade from these lignocellulosic materials. To utilize lignocellulosicbiomass for production of fuels and chemicals via fermentativeprocesses, it is necessary to convert the plant polysaccharides tosimpler sugars which are then fermented to products using a variety oforganisms. Direct hydrolysis of cellulose by mineral acids to monomersis possible at high temperature and pressure, leading to yield lossesdue to thermal decomposition of the sugars. One strategy to reduce theseyield losses is to use cellulases and potentially other enzymes todepolymerize the polysaccharides at moderate temperatures. Addition ofacid-stable thermotolerant hydrolytic enzymes such as cellulases andxylanases to the biomass slurry during the pretreatment allows the useof lower temperatures and pressures, as well as cheaper materials ofreactor construction, reducing both the capital and energy costs.Another approach is to combine the reduced severity pretreatment withenzymes together with fermentation under the same conditions, using asingle organism that produces the enzymes to degrade the material aswell as ferment the sugars to the value-added product of choice.

For commercially available enzymes to be used for this purpose, thepretreatment slurry must be neutralized and cooled to 40-50° C., addingsignificant cost to the process. Hence, it would be an improvement inthe art to degrade the soluble oligomers produced using acid,autohydrolysis or hot water washing pretreatments, at reduced severityand without neutralization using, for example, thermophilic and/oracidophilic enzymes.

Embodiments of the invention relate to a genetically modified organismcomprising at least one of a nucleic acid sequence and protein sequenceencoded by a nucleic acid sequence from Alicyclobacillus acidocaldarius.Such nucleic acids may include any of those nucleotide sequencesdescribed in the patent applications incorporated herein by reference.Such nucleic acids include, but are not limited to, those coding forenzymes capable of depolymerizing cellulosic polysaccharides to simplercarbohydrates and their transport into the bacterial cell and metabolismwithin the cell. Enzyme activities associated with transport into thecell and metabolism within the cell may be thermophilic and/oracidophilic in nature and general examples of nucleic acids coding forsimilar enzymes are described in the literature. Enzyme activitiesassociated with cellulose depolymerization and/or metabolism may bethermophilic (intracellular) and/or thermoacidophilic (extracellular)and include, without limitation, the following classes of enzymes:Alpha-glucosidases, Glucan 1,4-alpha-maltohydrolases, Glycosidases,Amylases, Acetyl esterases, Beta-galactosidases, Alpha amylases,Alpha-xylosidases, Cyclomaltodextrinases; Neopullulanases; Maltogenicalpha-amylases, Family 31 of glycosyl hydrolase,Alpha-L-arabinofuranosidases, Altronate hydrolases,poly-1,4-alpha-D-galacturonidase Xylan alpha-1,2-glucuronosidases,Cellulase/Endoglucanase, Polygalacturonases, Glycosyl hydrolases,Peptidoglycan hydrolases, N-acetylglucosaminidases, Endochitinases,Alpha-galactosidases, Endo-beta-1,4-mannanases, Cellobiosephosphorylases, Cyclic beta-1,2-glucan synthases, Glycogen debranchingenzymes, Acetyl hydrolases, Beta-1,4-xylanases, Beta-glucosidases,6-phospho-beta-glucosidases, Cinnamoyl ester hydrolases,Beta-glucuronidases, Xylan alpha-1,2-glucuronosidases,3-hydroxyisobutyryl-CoA hydrolases, Beta-glucosidases, glycosidases,Chitooligosaccharide deacetylases, glycosyl hydrolases (or glycosidehydrolases); esterases including acetylxylan esterases and p-cumaricacid esterases and ferulic acid esterases; and/or uronidases.

Additionally, embodiments of the invention relate in part to agenetically modified organism comprising, in addition to at least one ofthe nucleic acid sequences and protein sequences encoded by nucleicacids of Alicyclobacillus acidocaldarius, at least one of the nucleicacid sequences and protein sequences associated with fermenting sugarmolecules to products. Such nucleic acid sequences may code for proteinsequences which may be thermophilic and/or acidophilic in nature andgeneral examples of similar nucleic acids are described in theliterature.

The present invention also relates to isolated and/or purifiednucleotide sequences from the genome of Alicyclobacillus acidocaldariusselected from the sequences described in the patent applicationspreviously incorporated herein by reference.

Nucleotide, polynucleotide, or nucleic acid sequence will be understoodaccording to the present invention as meaning either double-stranded orsingle-stranded DNA and the transcription products of said DNAs.

As used herein, “fermentation” relates to the biological conversion of asugar molecule into a product. As used herein, a “product” is anychemical that can be made, at least in part, through a biologicalprocess. Examples of products include, but are not limited to, ethanol,acetic acid, glyoxylic acid, oxalic acid, lactic acid,3-hydroxypropionic acid, glycerol, 1,2-propanediol, 1,3-propanediol,propionic acid, acetone, fumaric acid, succinic acid, malic acid,butyric acid, 1-butanol, 2,3-butanediol, acetoin, aspartic acid,1,2-butanediol, itaconic acid, glutamic acid, citric acid, aconiticacid, cis-cis muconic acid, gluconic acid, kojic acid, amino acids,vitamins, alginate, cellulose, curdlan, chondroitin, cyanophycin,gellan, heparin, hyaluronic acid, poly-gamma-glutamic acid,poly-epsilon-lysine, polyhydroxyalkanoates, pullulan, scleroglucan,xanthan, indigo, and those chemicals set forth in the BREW report fromthe University of Utrect, (Patel, M. et al., (2006), “Medium andlong-term opportunities and risks of the biotechnological production ofbulk chemicals from renewable resources: The potential of whitebiotechnology”. The BREW Project. Final Report prepared under theEuropean Commission's GROWTH Programme (DG Research),(publica.fraunhofer.de/eprints/N-48834.pdf)), the entirety of thecontents of which are incorporated herein by this reference.

Although the following sections related to Hydrolysis AssociatedMolecules (HAMs) and Fermentation Associated Molecules (FAMs), thetechniques therein apply equally to all other nucleotide sequencesisolated and/or purified from the genome of acidophilic and orthermophilic organisms, such as, without limitation, Alicyclobacillusacidocaldarius and those HAMs and FAMs described in the patentapplications previously incorporated herein by reference.Expression/Integration of Hydrolysis Associated Molecules

In embodiments of the invention, one or more Hydrolysis AssociatedMolecules (HAMs) may be incorporated and/or inserted into an organismthat has the ability to ferment sugars to products. The HAMs may includeregulatory factors and/or nucleic acids coding for proteins associatedwith, involved in, and/or assisting in the breakdown and/or hydrolysisof biomass (i.e., polysaccharides, cellulose, lignocellulose,hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates,complex carbohydrates, chitin, heteroxylans, glycosides, xylan-,glucan-, galactan, or mannan-decorating groups, etc.) into more simplesugar molecules and/or sugar monomers. A non-exhaustive list of theseproteins and/or enzymes include: cellulases (i.e., endo and/or exocellulases such as endo-beta-1,4-glucanase or exo-beta-1,4-glucanase);hemicellulases (i.e., exo- and/or endo-beta-1,4-xylanase); one or morebeta-xylosidase enzymes; β-1,4-cellobiohydrolases (CBH I & CBH II);xylanases (XYN I & XYN II); β-glucosidase; α-L-arabinofuranosidase;acetyl xylan esterase; β-mannanase; and α-glucuronidase; esterases ofthe alpha-beta hydrolase superfamily; alpha beta hydrolase;alpha-glucosidases; alpha-xylosidase; alpha-L-arabinofuranosidase;altronate hydrolase; a cellulose/endoglucanase; a polygalacturonase; analpha-galactosidase; a cellobiose phosphorylase; a glycogen debranchingenzyme; an acetyl esterase/acetyl hydrolase; a beta-1,4-xylanase; acinnamoyl ester hydrolase; a carboxylesterase type B; a betagalactosidase/beta-glucuronidase; a xylan alpha-1,2-glucuronidase, a3-hydroxyisobutyryl-CoA hydrolase; a beta-glucosidase B-relatedglycosidase; and/or a chitooligosaccharide deacetylase and/or so forth.Additional proteins and/or enzymes may include ligninolytic enzymes suchas manganese peroxidase, lignin peroxidase, or the like.

Additionally, in embodiments, the HAMs may encompass all those nucleicacids and/or proteins associated with the replication, transcription,translation, and/or expression of nucleic acids involved with thebreakdown and/or hydrolysis of biomass and/or lignocellulose into moresimple sugar molecules and/or sugar monomers. In a non-limiting example,HAMs may comprise those nucleic acids involved with the regulation ofhydrolysis, such as, but not limited to, those nucleic acids and/orproteins that increase the replication, transcription, expression, etc.of HAMs. Further, HAMs may comprise those nucleic acids and/or proteinsthat are involved with the down-regulation of other HAMs. Anon-exhaustive list of these regulatory HAMs may include: transcriptionfactors, enhancers, repressors, DNA binding proteins, or the like.

In embodiments, the HAMs may include derivatives, analogs, and/ormodified versions of one or more HAMs. These derivative, analogs, and/ormodified versions may include those modifications carried out in vivo orin vitro. Some non-limiting examples of derivative, analogs, and/ormodifications include: substitutions, deletions, mutations,modifications etc. Modifications may comprise all modificationsoccurring to nucleic acids, DNA, RNA, proteins etc, such as but notlimited to acetylation, deacetylation, alkylation, methylation,demethylation, carboxylation, glycosylation, phosphorylation,hydroxylation, PEGylation, biotinylation, and/or any other type ofmodification known in the art.

In embodiments, the HAMs may encompass those nucleic acids, proteins,and/or enzymes that are derivatives of HAMs and include tags, markers,and/or other modifications. A non-exhaustive list of tags may include:His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltose bindingprotein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, or thelike. The HAMs may also includes those nucleic acids, proteins, and/orenzymes that include fluorescent markers, such as but not limited to,GFP, RFP, YFP, BFP, or the like.

In additional embodiments, HAMs may encompass nucleic acid or peptidesignals that direct secretion of HAM proteins from the cell in whichthey are produced. Examples of such signals include N-terminal and/orC-terminal sequences which direct localization and/or secretion of themolecules with which they are associated. In further embodiments, HAMsmay include transporter proteins, such as, by way of non-limitingexample, ABC transporter proteins which direct the secretion ofparticular molecules (such as other HAMs) to the extracellular orperiplasmic space. In embodiments, the signals may direct secretion of aHAM through any system for secretion, including, but not limited to,Endoplasmic Reticulum/Golgi Apparatus systems; vesicle mediatedsecretion systems; any of the Type I, II, III, IV, V, VI, and/or Tatsecretion systems; and/or Sec pathway systems.

The incorporation of the HAMs into the one or more organisms with theability to ferment sugars to products may enable the one or moreorganisms to convert cellulosic and/or other biomass compositions intoproducts in a variety pH and temperature conditions. In one embodiment,the one or more genetically modified organisms may carry out the biomassconversion in a range of pH conditions from at or less than a pH ofabout 7; at or less than a pH of about 5, about a pH of 1 to about a pHof 5; and/or from about a pH of 1 to about a pH of 1.3.

In embodiments, the ability of a genetically modified organism accordingto the present invention to function and/or convert biomass intoproducts in pH conditions of 1 to 5, may eliminate the need toneutralize and/or increase the pH to a range of about 5 to 6.0 forenzymatic hydrolysis and/or fermentation after dilute acid pretreatment,which, using conventional methods, is necessary for the hydrolysis andfermentation of cellulose to products. Indeed, in conventional methodsof dilute acid hydrolysis, the pretreatment hydrolysate needs to beneutralized to enable enzymatic hydrolysis and fermentation to occur. Assuch, in one embodiment, the HAMs may code for thermophilic,thermoacidophilic, and/or acidophilic enzymes, properties and/orcharacteristics, or in the alternative they may be derived from one ormore thermophilic, thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more of the HAM encodedproteins may function and/or have an optimum pH range of above a pH of5. Indeed, one or more of the HAMs may function and/or enable breakdownand/or hydrolysis of biomass and/or cellulose into more simple sugarmolecules and/or sugar monomers in pH conditions that range from a pH ofabout 5 to a pH of about 14.

In embodiments, one or more HAMs may function to and/or assist in thebreakdown and/or hydrolysis of biomass and/or cellulosic materials intomore simple sugar molecules and/or sugar monomers in a broad range oftemperatures. Conventional methods and techniques of pretreatment fordilute acid hydrolysis and low temperature acid hydrolysis occur attemperature ranges of about 160 degrees Celsius and a range of eighty(80) to one hundred (100) degrees Celsius, respectively. However, usingthe conventional methods, in order to begin the enzymatic hydrolysis andfermentation reactions, the pretreatment mixture/slurry needs to becooled to around forty (40) to fifty (50) degrees Celsius.

In contrast to the current and/or conventional methods, the HAMsincorporated into the organism may function to, assist in, and/or carryout the breakdown and/or hydrolysis of biomass and/or cellulosicmaterials into more simple sugar molecules and/or sugar monomers in abroad range of temperatures. Some non-limiting examples of temperatureranges include: at least about fifty (50) degrees Celsius; at leastabout seventy (70) degrees Celsius; from about forty five (45) degreesCelsius to about eighty (80) degrees Celsius; from about eighty (80)degree Celsius to about one hundred (100) degree Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In embodiments, the one or more HAMs may be isolated from, derived fromand/or originate from one or more organisms. The one or more organismsmay comprise the same organism, organisms within the same genus and/orwithin the same species, and/or different and/or distinct organisms. Inone non-limiting example, the HAMs are from one or more extremophilessuch as but not limited to: hyperthermophilic, thermophilic,acidophilic, thermoacidophilic, and/or polyextremophilic organisms. Somenon-limiting examples of organisms include: Alicyclobacillusacidocaldarius; Clostridium thermocellum; Clostridium thermolacticum;Clostridium thermohydrosulfuricum; Trichoderma reesei; a variety ofBacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter, B. pectinovorum, B. halodurans,etc.); Moorella thermoautotrophica; Moorella thermoacetica;Streptococcus (Lactobacillus) thermophilus; and/or other extremophilicbacteria that possess one or more nucleic acids associated with thebreakdown and/or hydrolysis of biomass and/or lignocellulosic materialinto more simple sugar molecules and/or sugar monomers. It is alsocontemplated that the HAMs may be isolated from, derived from, orotherwise originate from any type and/or kind of organism that includesone or more of the HAMs and/or other extremophilic bacteria that possessone or more nucleic acids encoding proteins involved in breakdown and/orhydrolysis of biomass and/or lignocellulosic material into products. Itis also contemplated that the HAMs may be isolated from, derived from,or otherwise originate from any type and/or kind of organism thatincludes one or more of the HAMs such as, but not limited to, thosefound at wzw.tum.de/mbiotec/cellmo.htm; or available from an institutionsuch as, but not limited to, the Advanced Biotechnology Center (ABC)(Italy); Interlab Cell Line Collection (Biotechnology Dept.) (Italy);the Australian Government Analytical Laboratories (AGAL); the AmericanType Culture Collection (ATCC); the Belgian Coordinated Collections ofMicroorganisms (BCCM); the Prime Minister's Services Federal Office forScientific, Technical and Cultural Affairs (OSTC) (Belgium); Bureau ofMicrobiology at Health Canada (BMHC); Centraalbureau voorSchimmelcultures (CBS) (the Netherlands); China Center for Type CultureCollection (CCTCC) (Wuhan); China Committee for Culture Collection ofMicroorganisms (Beijing); Colección Espa{umlaut over (n)}ola de CultivosTipo (CECT) (Spain); Collection Nationale De Cultures De Microorganismes(CNCM) (Institut Pasteur, France); Collection of Industrial Yeasts DBVPG(Perugia, Italy); Culture Collection of Algae and Protozoa (CCAP)(United Kingdom); Culture Collection of Yeasts (CCY) (Slovakia); CzechCollection of Microorganisms (CCM); Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ) (Germany); EuropeanCollection of Cell Cultures (ECACC) (United Kingdom); Institute ofAgriculture and Food Biotechnology (IAFB) Collection of IndustrialMicroorganisms (Poland); International Mycological Institute (IMI)(United Kingdom); International Patent Organism Depositary (IPOD)(Tsukuba, Japan); Korean Cell Line Research Foundation (KCLRF); KoreanCollection for Type Cultures (KCTC); Korean Culture Center ofMicroorganisms (KCCM); Microbial Strain Collection of Latvia (MSCL);National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC)(Bulgaria); National Collection of Agricultural and IndustrialMicroorganisms (NCAIM) (Budapest, Hungary); National Collection of TypeCultures (NCTC) (United Kingdom); National Collection of Yeast Cultures(NCYC); AFRC Institute of Food Research (United Kingdom); NationalCollections of Industrial, Food and Marine Bacteria (NCIMB) (Scotland);National Research Center of Antibiotics (Moscow); Polish Collection ofMicroorganisms (PCM) (Poland); Russian Collection of Microorganisms(VKM); and the Russian National Collection of Industrial Microorganisms(VKPM) (Russia).

In embodiments, one or more of the HAMs may be incorporated into,integrated into, and/or otherwise expressed in one or more organismsthat may already have the ability to ferment sugars to products. Suchorganisms may be any organism known in the art that has the ability toferment sugars to alcohols or other products. A non-exhaustive list oforganisms may include: Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter, B. pectinovorum, B. halodurans, etc.); Moorellathermoautotrophica; Moorella thermoacetica; Streptococcus(Lactobacillus) thermophilus; Saccharomyces cerevisiae (yeast),Zymomonas mobilis, Candida shehatae, E. coli, those found at any of theweb sites or institutions listed herein, and/or other organisms thatpossess one or more nucleic acids coding for proteins involved infermentation of sugars to products.

It is additionally contemplated that the organism able to ferment sugarsto products may be any organism that has nucleic acids encoding for oneor more proteins involved with the fermentation pathways, such as, butnot limited to, hexokinase I, hexokinase II, glucokinase,glucose-6-phosphate isomerase, phosphofructokinase, aldolase,triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase,3-phosphoglycerate kinase, phosphoglycerate mutase, enolase, enolase I,pyruvate decarboxylase, citrate synthase, aconitase, isocitratedehydrogenase, succinate dehydrogenase, fumarase, xylose reductase,xylitol dehydrogenase, xylulokinase, phosphoketolase, lactatedehydrogenase, acetyl-CoA-acetyl transferase, β-hydroxybutyryl-CoAdehydrogenase, crotonase, butyryl CoA dehydrogenase, phosphobutyrylase,butyrate kinase, and alcohol dehydrogenase. In embodiments of thepresent invention, the organism may already include those enzymes and/ornucleic acids encoding enzymes, necessary for fermenting sugars toproducts. Some non-limiting examples of these enzymes include:hexokinase I, hexokinase II, glucokinase, glucose-6-phosphate isomerase,phosphofructokinase, aldolase, triosephosphate isomerase,glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase,phosphoglycerate mutase, enolase, enolase I, pyruvate decarboxylase,citrate synthase, aconitase, isocitrate dehydrogenase, succinatedehydrogenase, fumarase, xylose reductase, xylitol dehydrogenase,xylulokinase, phosphoketolase, lactate dehydrogenase, acetyl-CoA-acetyltransferase, β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoAdehydrogenase, phosphobutyrylase, butyrate kinase, and alcoholdehydrogenase.

In embodiments, one or more of the HAMs may include and/or be expressedwith one or more regulatory elements and/or proteins, such as but notlimited to, promoters, enhancers, transcription factors, activators,silencers, and/or so forth. Non-limiting examples of such regulatoryelements may be found in the patent applications previously incorporatedherein by reference. In this manner, the transcription and/or expressionof one or more of the HAMs may be regulated and/or controlled. In anon-limiting example, the HAMs may include a promoter regionsubstantially similar to promoter regions of one or more of theorganism's Feimentation Associated Molecules (FAMs) (those nucleic acidsencoding proteins involved in the fermentation of sugars to products).In this manner, transcription and/or expression of one or more of theHAMs occur at substantially the same time, or substantially simultaneouswith, the transcription and/or expression of one or more of the FAMs. Itis also contemplated that one or more of the HAMs may be included and/orintegrated under the control of the same promoter and/or promoterregions of the FAMs in one or more of the organisms. Some non-limitingexamples of types of promoters contemplated include: constitutivepromoters, tissue specific or development stage promoters, induciblepromoters, and/or synthetic promoters. In one embodiment, the promotermay be a standard promoter used in expression vectors, such as, but notlimited to, the T7 & Sp6 Phage promoters, which promote the expressionof inserted nucleic acids in a phage type vector.

In embodiments, one or more of the HAMs may comprise and/or be includedin a delivery system that may deliver, incorporate, transfer, and/orassist in the delivery and/or expression of one or more of the HAMs intoone or more of the organisms able to break down and/or convert celluloseto more simple sugar molecules. The delivery system may comprise anymethod for incorporating DNA, nucleic acids and/or vectors in cellsknown in the art, such as but not limited to, transfection,electroporation, lipofection, transformation, gene guns, BiolisticParticle Delivery Systems, and/or so forth.

It is additionally contemplated that one or more HAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs) bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood, and in many instances vectors and/or insertion of nucleicacids into vectors are commercially available (i.e., through Empiregenomics at empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the HAMs may include selective markers, tags,or the like. Such selective markers tags, etc. may enable thedetermination of a successful delivery, integration, and/or expressionof a vector including one or more of the HAMs. Some non-limitingexamples of selective markers, tags, etc. include: antibioticresistance, amino acid/nutrient markers, color markers, fluorescentmarkers (i.e., GFP, RFP, etc.), His-tags, FLAG tags, Calmodulin-tags,HA-tags, Maltose binding protein-tags, Thioredoxin-tags, S-tags,Strep-tags, Nus-tags, and/or so forth.

In embodiments, it is contemplated that one or more of the HAMs may beintegrated with, incorporated into, and/or become part of the genomeand/or DNA of the host organism. Integrating one or more of the HAMsinto the host DNA may provide for better expression, replication tosubsequent host progeny, and/or protection from DNA degradation by thehost organism. Numerous methods and/or vectors for integrating,inserting, and/or incorporating a desired nucleic acid into a hostgenome are well known in the art. A non-exhaustive list of potentialvectors include: phage lambda (λ), adeno-associated virus (AAV),adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., A multi-purpose vector system . . . , Nucleic AcidsResearch, 2005, vol. 33, no. 4, which is incorporated by referenceherein.

Expression/Integration of Fermentation Associated Molecules

In one embodiment of the invention one or more Fermentation AssociatedMolecules (FAMs), encoding for proteins associated with, involved in,and/or assisting in the fermentation of sugars to products may beincorporated and/or inserted into a organism that has the ability tobreak down lignocellulosic materials to more simple sugar molecules. TheFAMs may include any nucleic acids, proteins and/or enzymes that areassociated with the fermentation of sugars to products. A non-exhaustivelist of these proteins and/or enzymes includes: hexokinase I, hexokinaseII, glucokinase, glucose-6-phosphate isomerase, phosphofructokinase,aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphatedehydrogenase, 3-phosphoglycerate kinase, phosphoglycerate mutase,enolase, enolase I, pyruvate decarboxylase, citrate synthase, aconitase,isocitrate dehydrogenase, succinate dehydrogenase, fumarase, xylosereductase, xylitol dehydrogenase, xylulokinase, phosphoketolase, lactatedehydrogenase, acetyl-CoA-acetyl transferase, β-hydroxybutyryl-CoAdehydrogenase, crotonase, butyryl CoA dehydrogenase, phosphobutyrylase,butyrate kinase, and alcohol dehydrogenase.

In additional embodiments, the FAMs encompass all those nucleic acidscoding for proteins associated with the replication, transcription,translation, and/or expression of nucleic acids associated with thefermentation of sugars to a product. In a non-limiting example, FAMs maycomprise those nucleic acids constituting either regulatory sequences orcoding for proteins involved with the regulation of FAMs, such as, butnot limited to, those nucleic acid sequences and/or proteins thatincrease replication, transcription, expression, etc. of FAMs. Further,FAMs may comprise those nucleic acids that are involved with thedown-regulation of FAMs, either directly or through the proteins theymay encode. A non-exhaustive list of proteins encoded by these FAMs mayinclude: transcription factors, enhancers, repressors, DNA bindingproteins, or the like.

In further embodiments, the FAMs may include derivatives, analogs,and/or modified versions of one or more FAMs. These derivatives,analogs, and/or modified versions may include those modificationscarried out either in vivo or in vitro. Some non-limiting examples ofderivatives, analogs, and/or modifications include: substitutions,deletions, mutations, modifications etc. Modifications may comprise allmodifications occurring to nucleic acids, DNA, RNA, proteins etc, suchas, but not limited to, acetylation, deacetylation, alkylation,methylation, demethylation, carboxylation, glycosylation,phosphorylation, hydroxylation, PEGylation, biotinylation, and/or anyother type of modification known in the art.

In yet other embodiments, the FAMs may encompass those nucleic acids,regulatory sequences, proteins, and/or enzymes which are encoded by theFAMs that are derivatives of FAMs and/or include tags, markers, and/orother modifications. A non-exhaustive list of tags may include:His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltose bindingprotein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, or thelike. The FAMs may also includes those nucleic acids, proteins, and/orenzymes that include fluorescent markers, such as, but not limited toGFP, RFP, YFP, BFP, or the like.

Advantageously, the incorporation of the FAMs into the one or moreorganisms with the ability to break down biomass to more simple sugarmolecules may enable the one or more genetically modified organisms toconvert lignocellulose and/or other biomass materials more efficientlyinto a product under a variety pH and/or temperature conditions. In oneembodiment, the one or more genetically modified organisms may carry outthe biomass conversion in a range of pH conditions from at or less thana pH of about 7; at or less than a pH of about 5, from about a pH of 1to about a pH of 5, and/or from about a pH of 1 to about a pH of 1.3.

Advantageously, in one embodiment, the ability of the geneticallymodified organism to function and/or convert biomass into products at pHconditions of 1 to 5, eliminates the need to neutralize and/or increasethe pH to a range of about 5 to 6.0 for enzymatic hydrolysis andfermentation, which, using conventional methods, is necessary for thehydrolysis and fermentation of cellulose to products. Indeed, inconventional methods of dilute acid hydrolysis, the pretreatmenthydrolysate needs to be neutralized to enable enzymatic hydrolysis andfermentation to occur. As such, in one embodiment, the FAMs may code forenzymes which comprise thermophilic, thermoacidophilic, and/oracidophilic properties and/or characteristics, or in the alternative maybe derived from, analogs of, and/or homologues of one or morethermophilic, thermoacidophilic, and/or acidophilic FAMs from otherorganisms.

It is additionally contemplated that one or more of the proteins encodedby the FAMs may function and/or have an optimum pH range of above a pHof 5. Indeed, one or more of the proteins encoded by the FAMs mayfunction and/or enable fermentation of sugar to products in pHconditions that range from a pH of about 5 to a pH of about 14.

In still another embodiment, enzymes encoded by one or more FAMs mayfunction to and/or assist in fermenting sugars to products in a broadrange of temperatures. Conventional methods and techniques ofpretreatment using dilute acid hydrolysis and low temperature acidhydrolysis occur at temperature ranges of about 160 degrees Celsius anda range of eighty (80) to one hundred (100) degrees Celsius,respectively. However, using the conventional methods, in order to beginthe enzymatic hydrolysis and fermentation reactions, the pretreatmentmixture/slurry needs to be cooled to around forty (40) to fifty (50)degrees Celsius.

In contrast to the current and/or conventional methods, the FAMsincorporated into one or more of the organisms described herein mayfunction to, assist in, and/or carry out the fermentation process in abroad range of temperatures. Some non-limiting examples of temperatureranges include: at least about fifty (50) degrees Celsius, at leastabout seventy (70) degrees Celsius, from about forty five (45) degreesCelsius to about eighty (80) degrees Celsius; from about eighty (80)degree Celsius to about one hundred (100) degree Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In further embodiments, the one or more FAMs may be isolated from,derived from and/or originate from one or more organisms. The one ormore organisms may comprise the same organism, organisms within the samegenus and/or within the same species, and/or different and/or distinctorganisms. In one non-limiting example, the FAMs are from one or moreextremophiles such as but not limited to: hyperthermophilic,thermophilic, acidophilic, thermoacidophilic, and/or polyextremophilicorganisms. Some non-limiting examples of organisms include:Alicyclobacillus acidocaldarius, Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter B. pectinovorum, B. halodurans, etc.); Moorellathermoautotrophica; Moorella thermoacetica; Streptococcus(Lactobacillus) thermophilus, those found at any of the web sites orinstitutions listed herein, and/or other organisms that possess one ormore nucleic acid sequences involved in fermentation of sugars toproducts. It is also contemplated that the FAMs may be isolated from,derived from, or otherwise originate from non-extremophilic organisms,such as but not limited to Saccharomyces cerevisiae (yeast), Zymomonasmobilis, Candida shehatae, E. coli, and/or other organisms which possessone or more nucleic acids involved in fermentation of sugars toproducts.

In embodiments, one or more of the FAMs may be incorporated into,integrated into, and/or otherwise expressed in one or more organismsthat already have the ability to break down lignocellulose and/orbiomass to more simple sugar molecules. Such organisms may be anyorganism known in the art that has the ability to break downlignocellulose to more simple sugar molecules. A non-exhaustive list ofsuch organisms may include: Alicyclobacillus acidocaldarius; Clostridiumthermocellum; Clostridium thermohydrosulfuricum; and/or any otherorganism that is known to have the ability to break down lignocelluloseand/or biomass. It is additionally contemplated that the organism ableto break down lignocellulose and/or biomass may be any organism that hasnucleic acid sequences encoding one or more types of endo and/or exocellulases, i.e., endo-beta-1,4-glucanase; one or more types ofhemicellulases, i.e., endo-beta-1,4-xylanase; and/or one or morexylosidase enzymes.

In one embodiment of the present invention, an organism may alreadyinclude enzymes and/or nucleic acids encoding enzymes, for breaking downat least one of the three major components of lignocellulose: cellulose,hemicellulose, and lignin. Some non-limiting examples of these enzymesinclude endo- and/or exo-β-1,4-glucanases. The endo- and/orexo-β-1,4-glucanases may function to hydrolyze the linked glucoseresidues (endo activity) and/or hydrolyze the ends of the cellulosepolymers (exo activity). Additionally, the organism may already includeendo-β-1,4-xylanase; and/or one or more xylosidase enzymes, such enzymespossessing the ability to hydrolyze one or more types of hemicellulosepolymers. In an additional embodiment, the organism may includeadditional and/or accessory enzymes, proteins, or nucleic acid sequencesthat may assist in completely hydrolyzing, transporting, metabolizingand/or breaking down the one or more types of lignocellulose polymersinto more simple sugar molecules and/or sugar monomers.

In embodiments, it is contemplated that the organism may include one ormore enzymes and/or nucleic acids encoding enzymes that have the abilityto break down, transport, metabolize, and/or hydrolyze lignin molecules;including nucleic acids encoding for ligninolytic enzymes such asmanganese peroxidase, laccase, lignin peroxidase, or the like.

In embodiments, one or more of the FAMs may include and/or be expressedwith one or more regulatory factors, regulatory elements, and/orproteins, such as, but not limited to, promoters, enhancers,transcription factors, activators, silencers, and/or so forth.Non-limiting examples of such regulatory elements may be found in thepatent applications previously incorporated herein by reference. In thismanner, the transcription and/or expression of one or more of the FAMsmay be regulated and/or controlled. In a non-limiting example, the FAMsmay include a promoter region substantially similar to promoter regionsof one or more of the organism's Hydrolysis Associated Molecules (HAMs)(those nucleic acids involved in breaking down lignocellulosic materialsto simpler sugars). In this manner, transcription and/or expression ofone or more of the FAMs occur at substantially the same time, orsubstantially simultaneous with, the transcription and/or expression ofthe Hydrolysis Associated Molecules. It is also contemplated one or moreof the FAMs may be included and/or integrated under the control of thesame promoter and/or promoter regions of the Hydrolysis AssociatedMolecules in one or more of the organisms. Some non-limiting examples oftypes of promoters contemplated include: constitutive promoters, tissuespecific or development stage promoters, inducible promoters, and/orsynthetic promoters. In one embodiment, the promoter may be a standardpromoter used in expression vectors, such as but not limited to, the T7& Sp6 Phage promoters, which promote the expression of inserted nucleicacids in a phage type vector.

In embodiments, one or more of the FAMs may comprise and/or be includedin a delivery system that may deliver, incorporate, transfer, and/orassist in the delivery and/or expression of one or more of the FAMs intoone or more of the organisms able to break down and/or convertlignocellulose to more simple sugar molecules. The delivery system maycomprise any method for incorporating DNA, nucleic acids and/or vectorsinto cells known in the art. A non-exhaustive list of methods mayinclude: transfection, electroporation, lipofection, transformation,gene guns, Biolistic Particle Delivery System, and/or so forth.

It is additionally contemplated that one or more FAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood in the art, and in many instances vectors and/or insertion ofnucleic acids into vectors are commercially available (i.e., throughEmpire genomics empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the FAMs may include and/or encode selectivemarkers, tags, or the like, that may enable the determination of asuccessful delivery, integration, and/or expression of a vectorincluding one or more of the FAMs. Some non-limiting examples ofselective markers, tags, etc. include: antibiotic resistance, aminoacid/nutrient markers, color markers, fluorescent markers (i.e., GFP,RFP, etc.), His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltosebinding protein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags,and so forth.

In embodiments, it is contemplated that one or more of the FAMs may beintegrated with, incorporated into, and/or become part of the genomeand/or DNA of the host organism. Integrating one or more of the FAMsinto the host DNA may provide for better expression, replication tosubsequent host progeny, and/or protection from DNA degradation by thehost organism. There are numerous methods and/or vectors forintegrating, inserting, and/or incorporating a desired nucleic acid intoa host genome which are well known in the art. A non-exhaustive list ofpotential vectors include: phage lambda (λ), adeno-associated virus(AAV), adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., A multi-purpose vector system . . . , Nucleic AcidsResearch, 2005, vol. 33, no. 4, which is incorporated by referenceherein.

BioProcessing

In embodiments, one of more of the genetically modified organismsdescribed herein may produce one or more proteins from the FAMs and/orHAMs intracellularly and/or extracellularly. In producing proteinsextracellularly, the genetically modified organism may excrete theproteins, enzymes, etc. into the extracellular and/or surroundingenvironment. It is additionally contemplated that the outer membraneand/or cell wall of the genetically modified organism may be coated withand/or include one or more proteins encoded by the HAMs. In this manner,breakdown and/or hydrolysis of biomass/lignocellulosic material isrealized in the extracellular environment.

In embodiments, one or more of the genetically modified organismsdescribed herein may convert biomass and/or lignocellulosic material toproducts at least partially intracellularly, or otherwise internal tothe genetically modified organism. In a non-limiting example, at leastpartially hydrolyzed sugar molecules may be brought and/or transportedinto the genetically modified organism and fermented to ethanol oranother product. The one or more genetically modified organism may thenexcrete the ethanol or other product into the extracellular environment.

It is contemplated that one more of the genetically modified organismsdescribed herein may have multiple methods of use, uses, and/orapplications in a wide variety of industries, laboratories, markets,etc.

In embodiments, one or more of the genetically modified organismsdescribed herein may be applied to biomass and/or lignocellulosicmaterial such that the biomass and/or lignocellulosic material is brokendown, depolymerized, and subsequently converted to products. The one ormore genetically modified organisms may be applied to the biomass orlignocellulosic material/composition in any manner known in the art. Ina non-limiting example, the biomass and/or lignocellulosic material maybe ground up and/or undergo a grinding process. Additionally, thebiomass and/or lignocellulosic material may undergo variouspretreatments prior to exposure to the recombinant organism. Suchtreatments may include, but are not limited to, sulfur dioxide, steamexplosion, acid hydrolysis, ammonia hydrolysis, autohydrolysis, or thelike. Additional, non-limiting examples are described in Thomas et al.,which is incorporated by reference herein.

In embodiments, one or more of the genetically modified organismsdescribed herein may be applied to biomass and/or lignocellulosicmaterial, such as, but not limited to, cellulose, through the use ofmultiple methods and/or procedures. In one non-limiting example, alignocellulosic composition may comprise one or more solidsubstrates/phases which are inoculated with the one or more geneticallymodified organisms, and/or cultures thereof. Additionally, thelignocellulosic composition may be embodied in a liquid, orsubstantially liquid substrate/phase; and the one or more geneticallymodified organisms introduced therein.

In embodiments, it is additionally contemplated that enzymes and/orproteins encoded by any and/or all of the HAMs and/or FAMs may beextracted, removed and/or isolated from one or more genetically modifiedorganisms described herein. These enzymatic and/or proteinaceouscompositions may then be applied to biomass and/or lignocellulosicmaterial.

In embodiments, the biomass and/or lignocellulosic composition isinoculated with a genetically modified organism culture. It iscontemplated there may be a variety of conditions (i.e., pH,concentrations of biomass and/or lignocellulosic material, concentrationof genetically modified organisms, temperature, pressure, etc.) at whichhydrolysis of the biomass and/or lignocellulosic material and subsequentfermentation may occur. Indeed, the pH of the hydrolysis may includeranges of at or less than a pH of about 7; at or less than a pH of about5; about a pH of 1 to about a pH of 5; and/or from about a pH of 1 toabout a pH of 1.3.

In embodiments, the ability of the one or more hydrolysis and/orfermentation reactions occurring in pH conditions of 1 to 5, mayeliminate the need to neutralize and/or increase the pH to a range ofabout 5 to 6 following acid pretreatment for enzymatic hydrolysis andfermentation that, using conventional methods, is necessary for thehydrolysis and fermentation of lignocellulosic material to products.Indeed, in conventional methods of dilute acid hydrolysis, thepretreatment hydrolysate needs to be neutralized to enable enzymatichydrolysis and fermentation to occur. As such, in one embodiment, theHAMs may code for enzymes that comprise thermophilic, thermoacidophilic,and/or acidophilic properties and/or characteristics, or in thealternative may be derived from one or more thermophilic,thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more hydrolysis and/orfermentation reactions may occur and/or have an optimum pH range ofabove a pH of 5. Indeed, the hydrolysis and/or fermentation reactionsmay occur in pH conditions that range from a pH of about 5 to a pH ofabout 14.

In embodiments, the one or more hydrolysis and/or fermentation reactionsmay occur in a broad range of temperatures. Some non-limiting examplesof temperature ranges include: from about fifty (50) degrees Celsius;from about seventy (70) degrees Celsius; from about eighty (80) degreesCelsius to about one hundred (100) degree Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In embodiments, one or more of the genetically modified organismsdescribed herein may be grown and/or cultured in accordance with knownmethods. Such genetically modified organism cultures may be fluidlyapplied and/or contacted with biomass and/or lignocellulosiccompositions, such as but not limited to, polysaccharides, cellulose,lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers,carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides,xylan-, glucan-, galactan, or mannan-decorating groups.

In embodiments, cellular and/or proteinaceous extracts of thegenetically modified organism cultures may be isolated. Protein and/orcellular isolations and extractions may be conducted throughconventional methods. The cellular and/or proteinaceous extracts may beapplied to and/or contacted with the biomass and/or lignocellulosiccompositions as described herein.

In embodiments, one or more of the genetically modified organisms and/orone or more the cellular and/or proteinaceous extracts described hereinmay be applied and/or fluidly contacted with biomass and/orlignocellulosic compositions under thermophilic, acidophilic, and/orthermoacidophilic conditions. In a non-limiting example, the temperaturemay be at or above about fifty (50) degrees and/or the pH at or belowabout pH 5 in a treatment mixture, slurry, apparatus or other area inwhich the fluid application occurs.

It is contemplated that a variety of conventional methods and/ortechniques may be used to create the one or more genetically modifiedorganisms described herein. One non-limiting method is described in U.S.Pat. No. 4,624,922, which is incorporated by reference herein.

Integration of Pretreatment, Biological Processing, and Product Recovery

Typically, the fermentative biological production of products frombiomass proceeds through four distinct steps: pretreatment, cellulolytichydrolysis, fermentation, and recovery. The pretreatment and recoverysteps remain distinct in typical process designs, while the cellulolytichydrolysis and fermentations steps can be separate steps or can becombined in various ways.

The pretreatment step is typically a physical or chemical pretreatment,such as an acid pretreatment, that effects a modification of the biomassto aid in further processing with cellulase enzymes. Typically, after asuitable period, the acid of the pretreatment is neutralized beforeinitiating cellulolytic hydrolysis. In the most basic processingconfiguration, cellulolytic hydrolysis is carried out using exogenouslyadded enzymes and is carried out separately from the fermentation step(separate hydrolysis and fermentation, or SHF). Glucose generated fromthe cellulose during cellulolytic hydrolysis, and pentose sugarsreleased during the pretreatment, are separately consumed by fermentingorganism(s) that are introduced to ferment the simple sugars intoethanol or another product of value. After fermentation, the product isrecovered in a recovery step.

Additional processing configurations are typically utilized in which thecellulolytic hydrolysis using exogenously added enzymes and fermentationsteps may be integrated. These include separate cellulolytic hydrolysisfollowed by co-fermentation of the pentose and hexose sugars (separatehydrolysis and co-fermentation, or SHCF), simultaneous cellulolytichydrolysis and glucose fermentation (simultaneous saccharification andfermentation, or SSF) with separate pentose fermentation, simultaneouscellulolytic hydrolysis and co-fermentation of the glucose and pentosesugars (simultaneous saccharification and co-fermentation, or SSCF).

A final processing configuration can be used in which the organismutilized to ferment glucose released during cellulolytic hydrolysisserves as the source of the cellulolytic enzymes. In this configuration,a physical and/or chemical pretreatment is utilized to improve theaction of the endogenously-produced cellulolytic enzymes on thecellulose. This processing configuration is referred to as ConsolidatedBioprocessing (CBP).

In embodiments of the invention, the above schemes may be modifiedthrough the use of enzymes and/or organisms active and stable atincreased temperatures and decreased pH to allow the combination ofpretreatment and/or product recovery with the conventional biologicalprocessing steps. In certain embodiments, the pretreatment may beconducted at lowered temperatures relative to existing dilute acidpretreatment technology and this step may be carried out enzymaticallyutilizing thermoacidophilic lignocellulose-degrading enzymes. Thealtered pretreatment and subsequent biological processing (cellulolytichydrolysis and fermentation) may be carried out sequentially or combinedtogether. The pretreatment and biological processing, may, in certainembodiments, be carried out by isolated enzymes and/or organisms for thecellulolytic hydrolysis and/or fermentation steps according to thepresent invention. The enzymes may be added exogenously or may beproduced by the fermentation organisms in a Combined Pretreatment andConsolidated Bioprocessing (CPBP) configuration. In certain embodiments,the thermoacidophilic enzymes and organisms may include Alicyclobacillusacidocaldarius, genetically modified organisms comprising one or morenucleotides sequences derived or isolated from Alicyclobacillusacidocaldarius, or by extracts and/or lysates comprising one or moreproteins produced by or derived from Alicyclobacillus acidocaldarius. Inembodiments, the pretreatment step may comprise an acid pretreatment;alkaline pretreatment; hydrothermal pretreatment; and/or anorganosolvent pretreatment.

In further embodiments, the acid or alkali added to or generated duringa pretreatment step may not need to be neutralized before hydrolysisand/or fermentation. In embodiments, the non-neutralization of thepretreatment conditions may lead to the decreased function or death ofany unwanted organisms present on the incoming biomass to be treated. Inembodiments, the non-neutralization of the pretreatment conditions mayresult in decreased byproducts or unwanted products as the function ofany unwanted organisms present on the incoming biomass to be treated maybe decreased.

In additional embodiments, one or more genetically modified organisms,HAMs, and/or FAMs according to the present invention may play a role inthe pretreatment, cellulolytic hydrolysis, and fermentation, or in anycombination thereof. For example, a genetically modified organismaccording to the present invention may carry out processes contributingto pretreatment and cellulolytic hydrolysis, and a second geneticallymodified organism according to the present invention may separately orsimultaneously carry out the fermentation. In another non-limitingexample, thermoacidophilic enzymes according to the present inventionmay carry out processes contributing to pretreatment, and one or moregenetically modified organism(s) according to the present invention maycarry out processes contributing to cellulolytic hydrolysis and/orfermentation. In a further non-limiting example, a single geneticallymodified organism(s) according to the present invention may carry outprocesses contributing to pretreatment, cellulolytic hydrolysis, andfermentation.

In embodiments, a reduced severity acid pretreatment step may becombined with, or occur sequentially with, the addition ofthermoacidophilic lignocellulose-degrading enzymes, and occur separatelyor concurrently with one or both of the cellulolytic hydrolysis andfermentation steps.

Expression/Integration of Hydrolysis Associated Molecules andFermentation Associated Molecules

In embodiments of the invention one or more Hydrolysis AssociatedMolecules (HAMs) and/or one or more Fermentation Associated Molecules(FAMs) may be incorporated and/or inserted into an organism.

The incorporation of the HAMs and FAMs into the one or more organismsmay enable the one or more organisms to convert lignocellulosic and/orother biomass materials into a product at a variety of pH andtemperature conditions. In one embodiment, the one or more geneticallymodified organisms may carry out the biomass conversion in a range of pHconditions from at or less than a pH of about 7; at or less than a pH ofabout 5: about a pH of 1 to about a pH of 5; and/or from about a pH of 1to about a pH of 1.3.

In embodiments, the ability of the organism(s) to function and/orconvert biomass into products in pH conditions of 1 to 5, may eliminatethe need to neutralize and/or increase the pH to a range of about 5 to 6for enzymatic hydrolysis and/or fermentation after dilute acidpretreatment, which, using conventional methods, is necessary for thehydrolysis and fermentation of cellulose to products. Indeed, inconventional methods of dilute acid hydrolysis, the pretreatmenthydrolysate needs to be neutralized to enable enzymatic hydrolysis andfermentation to occur. As such, in one embodiment, the HAMs and FAMs maycode for or be enzymes which possess thermophilic, thermoacidophilic,and/or acidophilic properties and/or characteristics, or in thealternative may be derived from one or more thermophilic,thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more of the HAMs and FAMsmay function and/or have an optimum pH range of above a pH of 5. Indeed,one or more of the HAMs and FAMs may function and/or enable breakdownand/or hydrolysis of biomass and/or lignocellulosic material intoproducts in pH conditions that range from a pH of about 5 to a pH ofabout 14.

Conventional methods and techniques of pretreatment for dilute acidhydrolysis and low temperature acid hydrolysis occur at temperatureranges of about 160 degrees Celsius and a range of eighty (80) to onehundred (100) degrees Celsius, respectively. However, using theconventional methods, in order to begin the enzymatic hydrolysis andfermentation reactions, the pretreatment mixture/slurry needs to becooled to around forty (40) to fifty (50) degrees Celsius.

In contrast to the current and/or conventional methods, the HAMs andFAMs may function to, assist in, and/or carry out the breakdown and/orhydrolysis of biomass and/or lignocellulose into products in a broadrange of temperatures. Some non-limiting examples of temperature rangesinclude: at least about fifty (50) degrees Celsius; at least aboutseventy (70) degrees Celsius; from about forty five (45) degrees Celsiusto about eighty (80) degrees Celsius; from about eighty (80) degreeCelsius to about one hundred (100) degree Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius).

In embodiments, the one or more HAMs and FAMs may be isolated from,derived from and/or originate from one or more organisms. The one ormore organisms may comprise the same organism, organisms within the samegenus and/or within the same species, and/or different and/or distinctorganisms. In one non-limiting example, the HAMs and FAMs are from oneor more extremophiles such as but not limited to: hyperthermophilic,thermophilic, acidophilic, thermoacidophilic, and/or polyextremophilicorganisms. Some non-limiting examples of organisms include:Alicyclobacillus acidocaldarius; Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; Trichoderma reesei; avariety of Bacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter B. pectinovorum, B. halodurans,etc.); Moorella thermoautotrophica; Moorella thermoacetica;Streptococcus (Lactobacillus) thermophilus; and/or other extremophilicorganisms that possess one or more nucleic acids encoding proteinsinvolved in breakdown and/or hydrolysis of biomass and/orlignocellulosic material and fermentation of sugars into products. It isalso contemplated that the HAMs and FAMs may be isolated from, derivedfrom, or otherwise originate from any type and/or kind of organism thatincludes one or more of the HAMs and FAMs such as, but not limited, tothose found at any of the web sites or institutions listed herein.

In embodiments, one or more of the HAMs and FAMs may be incorporatedinto, integrated into, and/or otherwise expressed in one or moreorganisms that do not have the ability to break down and/or hydrolyzebiomass and/or lignocellulose and/or ferment sugars to products. Suchorganisms may be any organism known in the art, such as but not limitedto E. coli, Thermus thermophilus, and/or any other suitable organismknown in the art.

In embodiments, one or more of the HAMs and FAMs may include and/or beexpressed with one or more regulatory elements and/or proteins, such asbut not limited to, promoters, enhancers, transcription factors,activators, silencers, and/or so forth. Non-limiting examples of suchregulatory factors may be found in the patent applications previouslyincorporated herein by reference. In this manner, the transcriptionand/or expression of one or more of the HAMs and FAMs may be regulatedand/or controlled. In a non-limiting example, the HAMs and FAMs mayinclude a promoter region substantially similar to promoter regions ofone or more of the organism's Fermentation Associated Molecules (thosenucleic acids involved in the fermentation of sugars to products). Inthis manner, transcription and/or expression of one or more of the HAMsand FAMs occur at substantially the same time, or substantiallysimultaneous with, the transcription and/or expression of the endogenousFAMs.

It is also contemplated that one or more of the HAMs and FAMs may beincluded and/or integrated under the control of the same promoter and/orpromoter regions in one or more of the organisms. Some non-limitingexamples of types of promoters contemplated include: constitutivepromoters, tissues specific or development stage promoters, induciblepromoters, and/or synthetic promoters. In one embodiment, the promotermay be a standard promoter used in expression vectors, such as but notlimited to, the T7 & Sp6 Phage promoters, which promote the expressionof inserted nucleic acids in a phage type vector.

In embodiments, one or more of the HAMs and FAMs may comprise and/or beincluded in a delivery system that may deliver, incorporate, transfer,and/or assist in the delivery and/or expression of one or more of theHAMs and FAMs into one or more organisms. The delivery system maycomprise any method for incorporating DNA, nucleic acids and/or vectorsin cells known in the art, such as but not limited to, transfection,electroporation, lipofection, transformation, gene guns, BiolisticParticle Delivery Systems, and/or so forth.

It is additionally contemplated that one or more HAMs and FAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: transposons, plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood in the art, and in many instances vectors and/or insertion ofnucleic acids into vectors are commercially available (i.e., throughEmpire genomics at empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the HAMs and FAMs may include selectivemarkers, tags, or the like. Such selective marker and/or tags may enablethe determination of successful delivery, integration, and/or expressionof a vector including one or more of the HAMs and FAMs. Somenon-limiting examples of selective markers, tags, etc. include:antibiotic resistance, amino acid/nutrient markers, color markers,fluorescent markers (i.e., GFP, RFP, etc.), His-tags, FLAG tags,Calmodulin-tags, HA-tags, Maltose binding protein-tags,Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, and/or so forth.

In embodiments, it is contemplated that one or more of the HAMs and FAMsmay be integrated with, incorporated into, and/or become part of thegenome and/or DNA of the host organism. Integrating one or more of theHAMs and FAMs into the host DNA may provide for better expression,replication to subsequent host progeny, and/or protection from DNAdegradation by the host organism. Numerous methods and/or vectors forintegrating, inserting, and/or incorporating a desired nucleic acid intoa host genome are well known in the art. A non-exhaustive list ofpotential vectors include: phage lambda (λ), adeno-associated virus(AAV), adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., 2005, A multi-purpose vector system . . . , NucleicAcids Research, vol. 33, no. 4, which is incorporated by referenceherein.

EXAMPLES Example 1 Delivery/Expression of Fermentation AssociatedMolecules in Alicyclobacillus acidocaldarius

Nucleic acids encoding enzymes involved in the fermentation of sugars(i.e., glucose, xylose, galactose, arabinose, mannose, etc.) to productsare provided to and subsequently expressed in Alicyclobacillusacidocaldarius using conventional molecular cloning techniques. Nucleicacids to be provided include those nucleic acids encoding for theproteins involved in the pathways that convert sugars to a product(FAMs). A non-exhaustive list of these proteins include: hexokinase I,hexokinase II, glucokinase, glucose-6-phosphate isomerase,phosphofructokinase, aldolase, triosephosphate isomerase,glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase,phosphoglycerate mutase, enolase, enolase I, pyruvate decarboxylase,citrate synthase, aconitase, isocitrate dehydrogenase, succinatedehydrogenase, fumarase, xylose reductase, xylitol dehydrogenase,xylulokinase, phosphoketolase, lactate dehydrogenase, acetyl-CoA-acetyltransferase, β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoAdehydrogenase, phosphobutyrylase, butyrate kinase, and alcoholdehydrogenase. The FAMs may comprise nucleic acids from a wide varietyof organisms that use fermentation pathways. Some non-limiting examplesof these organisms include: Saccharomyces cerevisiae (yeast);Clostridium thermocellum; Clostridium thermohydrosulfuricum; a varietyof Bacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter, B. pectinovorum, B. halodurans,and/or so forth); Alicyclobacillus acidocaldarius; Clostridiumthermolacticum; Trichoderma reesei; Moorella thermoautotrophica;Moorella thermoacetica; Streptococcus (Lactobacillus) thermophilus;and/or those found at any of the web sites or institutions listedherein.

Example 1(a) Delivery of FAMs Via a Transpososome

A transpososome is constructed to include nucleic acids encodingproteins involved in fermentation of sugars, or FAMs. Included in thetranspososome are the nucleic acids necessary for insertion of thetransposon, copies of the MuA transposase, Mu transposon ends, andprovision for expression of the FAMs included in the transpososome inthe particular organism in which they are to be utilized. Transpososomesmay also be constructed using sequences and elements optimized for usein gram-positive bacteria. See, Pajunen et al., Microbiology (2005) 151,1209-1218. Conventional techniques may be used to construct thetranspososome. Additionally, the construction and/or insertion ofdesired nucleic acids into transpososomes is well known in the art, see,Pajunen et al. It is contemplated that multiple transpososomes may beconstructed with FAMs and may include a variety of promoters, enhancersthat may function to enhance and/or regulate expression of FAMs.

Once constructed, the transpososomes are introduced intoAlicyclobacillus acidocaldarius. Transpososome constructs may beintroduced into A. acidocaldarius by electroporation, and so forth. Suchtechniques are known in the art and are detailed in Sambrook J C,Fritsch R, Maniatas T 1989. Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, N.Y., p. 6.3-6.34; or Sambrookand Russell, Molecular Cloning: A Laboratory Manual (3-Volume Set),3^(rd) Ed. (available online at molecularcloning.com/); both referencesincorporated by reference herein.

Example 2 Delivery/Expression of Hydrolysis Associated Molecules into anOrganism Able to Ferment Sugar to Ethanol

Nucleic acids encoding enzymes involved in the hydrolysis, or breakdown,of lignocellulosic materials (i.e., biomass, cellulose, hemicellulose,etc.) to simpler sugars (Hydrolysis Associated Molecules (HAMs)) aregenetically introduced by conventional molecular cloning techniques intoorganisms that ferment sugars to ethanol and subsequently expressedtherein. HAMs to be provided include those nucleic acids coding for theproteins involved in the pathways that break down or hydrolyzecellulose, hemicellulose, etc. to more simple sugar molecules. Anon-exhaustive list of these proteins include: esterases of thealpha-beta hydrolase superfamily; alpha beta hydrolase;alpha-glucosidases; alpha-xylosidase; alpha-L-arabinofuranosidase;altronate hydrolase; a cellulose/endoglucanase; acellulase/endoglucanase; a polygalacturonase; an alpha-galactosidase; acellobiose phosphorylase; a glycogen debranching enzyme; an acetylesterase/acetyl hydrolase; a beta-1,4-xylanase; a cinnamoyl esterhydrolase; a carboxylesterase type B; a betagalactosidase/beta-glucuronidase; a xylan alpha-1,2-glucuronidase; a3-hydroxyisobutyryl-CoA hydrolase; a beta-glucosidase B-relatedglycosidase; and/or a chitooligosaccharide deacetylase, or as describedherein. The HAMs may comprise nucleic acids from a wide variety oforganisms that are able to hydrolyze and/or break down lignocellulosicmaterial to more simple sugar molecules.

Example 2(a) Delivery of HAMs Via a Transpososome

A transpososome is constructed to include nucleic acids encodingproteins involved in hydrolysis or breakdown of lignocellulosicpolymers, or HAMs. Included in the transpososome are the nucleic acidsnecessary for insertion of the transposon, copies of the MuAtransposase, Mu transposon ends, and provision for expression of theHAMs included in the transpososome in the particular organism in whichthey are to be utilized. Transpososomes may also be constructed usingsequences and elements optimized for use in gram-positive bacteria. See,Pajunen et al., Microbiology (2005) 151, 1209-1218. Conventionaltechniques may be used to construct the transpososome. Additionally, theconstruction and/or insertion of desired nucleic acids intotranspososomes is well known in the art, see, Pajunen et al. It iscontemplated that multiple transpososomes may be constructed with HAMsand may include a variety of promoters, enhancers that may function toenhance and/or regulate expression of HAMs.

Once constructed, the transpososomes are introduced into an organismthat is able to ferment and/or convert sugar molecules to a product. Anon-exhaustive list of potential organisms include: Clostridiumthermocellum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter, B. pectinovorum, B. halodurans, and/or so forth);and/or those described herein. The transpososome HAM constructs may beintroduced into the organism by any technique known in the art, such as,but not limited to, electroporation, and so forth. Such techniques areknown in the art and are detailed in Sambrook J C, Fritsch R, Maniatas T1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, N.Y, p. 6.3-6.34; or Sambrook and Russell,Molecular Cloning: A Laboratory Manual (3-Volume Set), 3^(rd) Ed.(available online at worldwideweb.molecularcloning.com/), bothreferences incorporated by reference herein.

Example 2(b) Recombination/Integration of Hydrolysis AssociatedMolecules into Genome of Organism Able to Ferment Sugar to Ethanol

One or more HAMs are integrated into the genome of an organism that isable to ferment and/or convert sugar molecules to a product. Atranspososome is made that includes the HAMs and contains sequences forpromoting the integration of the included HAM(s) into the genome. Thetranspososome construct may be any type and/or kind of transpososomeconstruct known in the art, such that the transpososome constructassists in the integration of the one or more HAMs into a host genome.Such transpososome may include Mu based transpososomes or the like.

Example 3 Transformation/Expression of Hydrolysis Associated Moleculesand Fermentation Associated Molecules in a Organism

Both HAMs and FAMs are expressed in a suitable organism. The HAMs andFAMs to be expressed include any and/or all of the Hydrolysis orFermentation Associated Molecules as described herein.

Example 3(a) Transformation of Hydrolysis Associated Molecules andFermentation Associated Molecules Using a Transpososome

One or more transpososomes are constructed including HAMs and/or FAMs.Similar to Example 1 and Example 2, included in each of thetranspososomes are the nucleic acids necessary for insertion andexpression of the nucleic acids in the transpososome. Included in thetranspososome are the nucleic acids necessary for insertion of thetransposon, copies of the MuA transposase, Mu transposon ends, andprovision for expression of the nucleic acids contained in thetranspososome in the particular organism in which they are to beutilized. Transpososomes may also be constructed using sequences andelements optimized for use in gram-positive bacteria. See, Pajunen etal., Microbiology (2005) 151, 1209-1218. Conventional techniques may beused to construct the transpososome. Additionally, the constructionand/or insertion of desired nucleic acids into transpososomes is wellknown in the art, see, Pajunen et al. It is contemplated that multipletranspososomes may be constructed with the HAMs and FAMs, and mayinclude a variety of promoters and/or enhancers that may function toenhance expression of HAMs and/or FAMs.

Once constructed, the transpososomes are introduced into a targetorganism. Some non-limiting examples of potential organisms include E.coli, any type and/or kind of thermophilic bacteria (e.g., Sulfolobus,Thermoproteus, etc.), and/or so forth. The transpososome constructs maybe introduced into the organism by any technique known in the art, or asdescribed herein.

Example 3(b) Integration of Hydrolysis and Fermentation AssociatedMolecules into Genome of Organisms

One or more FAMs and/or HAMs are integrated into the genome of A.acidocaldarius. A transpososome is made that includes FAMs and/or HAMsand contains sequences for promoting the integration of the includedFAMs and/or HAMs into the genome. The transpososome may be any typeand/or kind of transpososome construct known in the art, such that thetranspososome assists in the integration of the one or more FAMs and/orHAMs into a host genome. Such transpososomes may include Mu basedtranspososomes or the like.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims and their legal equivalents.

BIBLIOGRAPHIC REFERENCES

-   Garrote, G, H Dominguez, and J C Parajo, 2001, Manufacture of    xylose-based fermentation media from corncobs by posthydrolysis of    autohydrolysis liquors, Appl. Biochem. Biotechnol., 95:195-207.-   Hamelinck, C N, G van Hooijdonk, and A P C Faaij, 2005, Ethanol from    lignocellulosic biomass: techno-economic performance in short-,    middle-, and long-term, Biomass Bioenergy, 28:384-410.-   Laitinen et al., 2005, A Multipurpose Vector system for the    screening of libraries in bacteria, insect and mammalian cells and    expression in vivo, Nucleic Acids Research 33(4)-   Liu C, and C E Wyman, 2003, The effect of flow rate of compressed    hot water on xylan, lignin, and total mass removal from corn stover,    Ind. Eng. Chem. Res., 42:5409-5416.-   Lynd et al., 2002, Micro. and Mol. Biol. Rev., Vol. 66, No. 3, P.    506-577.-   Sambrook J C, Fritsch R, Maniatas T 1989. Molecular Cloning: A    Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,    N.Y, p. 6.3-6.34.-   Sambrook and Russell, 2001, Molecular Cloning: A Laboratory Manual    (3-Volume Set), 3^(rd) Ed. (available online at    molecularcloning.com/).-   Ng et al., 1981, Applied and Environmental Microbiology,    41(6):1337-1343.-   Tsao, G T, M R Ladisch, and H R Bungay, 1987. Biomass Refining, In    Advanced Biochemical Engineering, Wiley Interscience, N.Y., p.    79-101.

What is claimed is:
 1. A genetically modified bacterial or fungalorganism, the genetically modified organism comprising: at least oneexogenous nucleic acid isolated from Alicyclobacillus acidocaldarius andencoding a polypeptide having at least 90% homology to SEQ ID NO:69 andwherein the peptide has alpha-L-arabinofuranosidase activity activity;and at least one nucleic acid sequence encoding a polypeptide associatedwith fermenting sugar molecules to a product.
 2. The geneticallymodified organism of claim 1, wherein the at least one nucleic acid fromAlicyclobacillus acidocaldarius is integrated into the host organism'sgenome.
 3. The genetically modified organism of claim 1, wherein thepolypeptide has alpha-L-arabinofuranosidase activity at or above aboutfifty degrees Celsius.
 4. The genetically modified organism of claim 1,wherein the polypeptide comprises SEQ ID NO:
 69. 5. The geneticallymodified organism of claim 1, wherein the nucleic acid isolated fromAlicyclobacillus acidocaldarius has at least 90% homology to SEQ ID NO:68.
 6. The genetically modified organism of claim 1, wherein the nucleicacid isolated from Alicyclobacillus acidocaldarius is SEQ ID NO:
 68. 7.An extract isolated from the genetically modified organism of claim 1.8. A method for at least partially processing hemicellulose into aproduct, the method comprising: contacting a liquid culture medium ofthe genetically modified organism of claim 1 with hemicellulose.
 9. Themethod according to claim 8, wherein the contacting occurs at or aboveabout fifty degrees Celsius.
 10. A method for at least partiallyprocessing hemicellulose, the method comprising: isolating an enzymeextract from the genetically modified organism of claim 1; andcontacting the extract with hemicellulose.
 11. A method of producing aproduct from biomass, the method comprising: pretreating the biomasswith an acid; enzymatically hydrolyzing cellulose and hemicellulose inthe biomass to more simple sugars and concurrently fermenting the moresimple sugars to a product; and recovering the product; wherein at leasta portion of the hydrolyzing of cellulose and hemicellulose or thefermentation is performed by the genetically modified organism ofclaim
 1. 12. A method of producing a product from biomass, the methodcomprising: treating the biomass with an acid; enzymatically hydrolyzingcellulose and hemicellulose in the biomass to more simple sugars;fermenting the more simple sugars to a product; and recovering theproduct; wherein at least a portion of the hydrolyzing of cellulose andhemicellulose or the fermentation is performed by the geneticallymodified organism according to claim 1; and wherein the pretreatment,the hydrolyzing of cellulose and hemicellulose, and the fermentation areperformed concurrently.
 13. A method of processing biomass, the methodcomprising: pretreating the biomass with an acid and concurrentlyenzymatically hydrolyzing cellulose and hemicellulose in the biomass tomore simple sugars; wherein at least a portion of the hydrolyzing ofcellulose and hemicellulose is performed by the genetically modifiedorganism of claim 1.